Chinese Journal of

Natural Chinese Journal of Natural Medicines 2014, 12(6): 0415−0422 Medicines

doi: 10.3724/SP.J.1009.2014.00415

Antioxidant activity profiling by spectrophotometric methods of aqueous methanolic extracts of Helichrysum stoechas subsp. ru- pestre and Phagnalon saxatile subsp. saxatile

,Tarik Mohammed Chaouche 1, Riadh Ksouri 2, Faten Medini 2 ,٭Farah Haddouchi 1 Fatima Zohra Sekkal 3, Abdelhafid Benmansour 1

1 Laboratory of Natural Products, Department of Biology, Faculty of Sciences, Abou Bekr Belkaïd University, B. P 119, Tlemcen, 13000, Algeria; 2 Laboratory of Plant Adaptation to Abiotic Stresses, Biotechnologic Center in Borj-Cedria Technopol (CBBC), B. P 901, 2050 Hammam-Lif, Tunisia; 3 Department of Biotechnology, Faculty of Natural Science and Life, Abdelel Hamid Ibn Badiss University, Mostaganem, 27000, Algeria Available online 20 June 2014

[ABSTRACT] AIM: The aqueous methanolic extracts of two plants from Algeria, Helichrysum stoechas subsp. rupestre and Phagnalon saxatile subsp. saxatile, were investigated for their antioxidant activity. METHOD: Total phenolics, flavonoids, and tannins were determined by spectrophotometric techniques. .In vitro antioxidant and radical scavenging profiling was determined by spectrophotometric methods, through: Total antioxidant capacity, and radical scavenging effects by the DPPH and ABTS methods, reducing and chelating power, and blanching inhibition of the β-carotene. RESULTS: All of the extracts showed interesting antioxidant and radical scavenging activity. The highest contents in phenolics, tannins, and the highest total antioxidant capacity as gallic acid equivalents of 97.5 ± 0.33 mg GAE/g DW was obtained for the flowers of H. stoechas subsp. rupestre extract in the phosphomolybdenum assay. An extract of the leafy stems of P. saxatile subsp. saxatile revealed the highest content of flavonoids, and the highest antioxidant activity by the radical scavenging and β-carotene

assays when compared with standards. The best activity was by the scavenging radical DPPH with an IC50 value of 5.65 ± 0.10 µg·mL−1. CONCLUSION: The studied medicinal plants could provide scientific evidence for some traditional uses in the treatment of dis- eases related to the production of reactive oxygen species (ROS) and oxidative stress.

[KEY WORDS] Helichrysum stoechas subsp. rupestre; Phagnalon saxatile subsp. saxatile; Phenolic compounds; Antioxidant activity [CLC Number] R965 [Document code] A [Article ID] 2095-6975(2014)06-0415-08

In order to prolong the storage stability of foods, and to re- Introduction duce damage to the human body, synthetic antioxidants, such When oxygen is supplied in excess, or its reduction is as butylated hydroxyanisole (BHA) and butylated hydroxyto- insufficient, reactive oxygen species (ROS) are generated. It luene (BHT), are commonly used. However, restrictions on is generally accepted that free radicals play an important role the use of these compounds are being imposed because of in the development of tissue damage and pathological events [1]. their carcinogenicity and some side effects [2]. Thus, evalua- tion of the antioxidant activity of naturally occurring sub- stances has been the focus of interest in recent years [3-4]. The [Received on] 20-Mar.-2013 antioxidant activity of plants, which contain a diverse group [*Corresponding author] Farah Haddouchi: Tel: 213-772735216, E-mail: [email protected] of phenolic compounds, is mainly due to their redox proper- These authors have no conflict of interest to declare. ties which make them act as reducing agents, hydrogen do-

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nors, and singlet oxygen quenchers. They also may have a To each sample extract (100 µL) suitably diluted, Na2CO3 (2 metal chelating potential [5]. mL, 2%, W/V) was added, and the mixture shaken and The Helichrysum genus (Asteraceae) includes more than allowed to stand for 5 min. Folin–Ciocalteu reagent (100 500 species that are widespread around the world. A great µL, 1 mol·L−1) was added. After incubation for 30 min at number of biological activities are usually attributed to this room temperature in the dark, the absorbance versus a genus, such as anti-inflammatory, anti-allergic, antioxidant, prepared blank was read at 750 nm. Total phenolic content antimicrobial, cough relief, and treatment of colds and wounds was expressed as mg gallic acid equivalents per gram of [6]. The chemistry of the Helichrysum genus is complex, with dry weight (mg GAE/g DW) using a calibration curve for a wide variety of chemical classes, among which are flavono- gallic acid (0–400 µg·mL−1) ids, chalcones, phloroglucinol derivatives, essential oils, Total flavonoids α-pyrones, and diterpenes [7]. The genus Phagnalon (Astera- Measurement of flavonoid concentration in different ceae) is represented by about thirty species distributed aqueous methanolic extracts was based on the method de- worldwide, six of which are typical of the Mediterranean scribed by Dewanto et al [15]. An aliquot (250 µL) of the sam- [8] region . Different Phagnalon species are used in traditional ples was added to test tubes containing 5% NaNO2 solution medicine, and a variety of extracts have been examined [9]. (75 µL), and mixed for six min. Then, freshly prepared 10%

Phytochemical investigations on Phagnalon species are AlCl3 solution (150 µL) was added, and after 5 min at room mainly devoted to the study of P. rupestre, and report the temperature, 1 mol·L−1 NaOH (0.5 mL) was added. The final presence of terpenoids [10], flavonoids [11], hydroquinone gly- volume was adjusted to 2.5 mL with distilled water and tho- cosides, and caffeoylquinic acid derivatives [12]. A survey of roughly mixed. The absorbance of the mixture was deter- the literature shows the presence in the aerial parts of P. sa x - mined at 510 nm against the same mixture without the sample atile of an essential oil [13] and flavonoids [10]. as a blank. Total flavonoid content was expressed as mg ca- However, scientific information on the antioxidant prop- techin per gram of dry weight (mg CE/g DW), through the erties of various plants, particularly those that are less widely calibration curve of (+)-catechin (0–400 μg·mL−1). used in the culinary arts and medicine is still rather scarce. Total condensed tannins There are any reports of the Helichrysum and Phagnalon The content of condensed tannins were determined by the species belonging to Algerian flora. The aim of the present procedure of Sun et al [16]. Fifty microliters of different work is to study the phenolic content and antioxidant activity aqueous methanolic extracts was mixed with 4% vanil- of aqueous methanolic extracts of Helichrysum stoechas lin-methanol solution (3 mL) and 1 mol·L−1 hydrochloric acid subsp. rupestre auct. and Phagnalon saxatile (L.) Cass. subsp. (1.5 mL). The mixture was allowed to stand for 15 min and saxatile, collected from Algeria. the absorbance was measured at 500 nm. The amount of total condensed tannins is expressed as mg catechin per gram of Material and Methods dry weight (mg CE/g DW). The calibration curve range of Preparation of plant extracts (+)-catechin was 0–400 μg·mL−1. The flowering aerial parts of the plants were collected in Determination of antioxidant activities March 2011, from two different regions of Tlemcen in the Total antioxidant capacity west of Algeria. H. stoechas subsp. rupestre was collected The assay is based on the reduction of Mo (VI) to Mo (V) from Honaine (35°10′35″ N, 1°39′18″ W) and P. saxatile subsp. by the extract and subsequent formation of a green phos- saxatile from Fellaoucen (35°02′06″ N, 1°36′21″ W). They phate/Mo (V) complex at acidic pH [17]. An aliquot (0.1 mL) were identified in the Laboratory of Natural Products, De- of aqueous methanolic extract was combined to 1 mL of rea- partment of Biology, University of Tlemcen, Algeria. Voucher gent solution (0.6 mol·L−1 sulfuric acid, 28 mmol·L−1 sodium specimens were deposited at the Herbarium of the Laboratory. phosphate and 4 mmol·L−1 ammonium molybdate). The tubes The plants were dried at room temperature for two weeks. were incubated in a thermal block at 95 °C for 90 min. After The flowers were separated from the leafy stems and the ex- the mixture had cooled to room temperature; the absorbance tracts were obtained separately by the magnetic stirring for 24 of each solution was measured at 695 nm against a blank. The h of the plant powder (2 g) with aqueous methanol 80 : 20 (25 antioxidant capacity was expressed as mg gallic acid equiva- mL). The aqueous methanolic extracts were evaporated at 45 lent per gram dry weight (mg GAE/g DW). The calibration °C under reduced pressure, re-dissolved in methanol at a curve range was 0–500 μg·mL−1. concentration of 10 mg·mL−1, and stored at 4 °C for further DPPH assay use. The antioxidant activity of the aqueous methanolic ex- Quantification of phenolic classes tracts of the plants was measured using the stable DPPH me- Total polyphenols thod based on the reduction of an alcoholic DPPH solution in The amounts of total phenolics in the aqueous methanolic the presence of a hydrogen donating antioxidant due to the extracts were determined with the Folin–Ciocalteu reagent formation of the non-radical form DPPH-H by the reaction [18]. using the method described by Vermerris and Nicholson [14]. The sample was diluted in pure extraction solvent at different

– 416 – Farah Haddouchi, et al. / Chin J Nat Med, 2014, 12(6): 415−422 concentrations (0–3 mg·mL−1), then each diluted plant extract sessed as described by Zhao et al [21]. Different concentrations −5 −1 (50 μL) was added to a 6.34 × 10 mol·L DPPH methanolic of plant extracts were added to FeCl2 (4H2O) solution (50 µL, solution (1 950 μL). The mixture of different extract concen- 2 mmol·L−1) and left for incubation at room temperature for 5 tration and DPPH was placed in the dark at room temperature min. Then, the reaction was initiated by adding ferrozine (0.1 for 30 min. The absorbance of the resulting solution was then mL, 5 mmol·L−1), and the mixture was adjusted to 3 mL with read at 517 nm. BHT was used as a positive control. The abil- distilled water, shaken vigorously, and left standing at room ity to scavenge the DPPH radical was calculated using the temperature for 10 min. The absorbance of the solution was following equation: measured at 562 nm. The percentage of inhibition of ferro- 2+ DPPH scavenging effect (%) = [A0 – A1/A0] × 100 (1) zine-Fe complex formation was calculated using the formu-

where A0 is the absorbance of the control at 30 min, and la given below: A1 is the absorbance of the sample at 30 min. The antiradical Metal chelating effect (%) = [A0 – A1/A0] × 100 activity was expressed as the concentration of an antioxidant where A0 and A1 have the same meaning as in Eq. (1). −1 −1 . (µg·mL ) needed to decrease the initial DPPH concentration The results were expressed as IC50 values (mg·mL ) by 50% (IC50). β-Carotene bleaching test Iron reducing power A slightly modified Koleva et al [22] method was em- The capacity of the plant extracts to reduce Fe3+ was as- ployed to estimate the aqueous methanolic extracts capacity sessed by the method of Oyaizu [19]. In the reducing power to inhibit β-carotene bleaching. Two milligrams of assay, the presence of antioxidants in the sample would result β-carotene was dissolved in chloroform (20 mL) and to this in the reduction of Fe3+ to Fe2+ by donating an electron. solution (4 mL), linoleic acid (40 mg) and Tween 40 (400 Aqueous methanolic extract (1 mL) was mixed with sodium mg) were added. The chloroform was evaporated under phosphate buffer (2.5 mL, 0.2 mol·L−1, pH 6.6) and 1% potas- vacuum at 40 °C and oxygenated water (100 mL) was added, sium ferricyanide (2.5 mL), and the mixture was incubated at then the fresh emulsion was vigorously shaken. An aliquot 50 °C for 20 min. After that, 10% trichloroacetic acid (2.5 mL) (150 µL) of the β-carotene/linoleic acid emulsion was dis- was added, and the mixture was centrifuged at 650 × g for 10 tributed in 96-well microtitre plates and aqueous methanolic min. The upper layer fraction (2.5 mL) was mixed with dis- solutions of the test samples (10 µL) or authentic standard tilled water (2.5 mL) and 0.5 mL of ferric chloride (0.5 mL) (BHA) were added. The microtitre plates were incubated at and thoroughly mixed. The absorbance was measured at 700 50 °C for 120 min, and the absorbance was measured using nm. Ascorbic acid and BHT were used as a positive control. A a model EAR 400 microtitre reader at 470 nm. Readings of higher absorbance indicates a higher reducing power. The all samples were performed immediately (t = 0 min) and −1 EC50 value (µg·mL ) is the effective concentration giving an after 120 min of incubation. The antioxidant activity of the absorbance of 0.5 for reducing power, and was obtained from extracts was evaluated in terms of bleaching inhibition of linear regression analysis. the β-carotene using formula: ABTS assay β-Carotene bleaching inhibition (%) = [S−C120/C0 −C120] × The ABTS radical-scavenging activity of extracts was 100 determined according to Re et al [20]. The ABTS·+ cation radical where C0 and C120 are the absorbance values of the con- −1 ·+ was produced by the reaction between 14 mmol·L ABTS trol at 0 and 120 min, respectively, and S is the sample absor- solution (5 mL) and 4.9 mmol·L−1 potassium persulfate (K S O ) 2 2 8 bance at 120 min. The results were expressed as IC50 values solution (5 mL), stored in the dark at room temperature for 16 h. (µg·mL−1). Before use, this solution was diluted with ethanol to obtain an Correlation analysis absorbance of 0.700 ± 0.020 at 734 nm. In a final volume of 1 Values shown in the Tables were x ± s of three parallel mL, the reaction mixture comprised ABTS·+ solution (950 µL) measurements. The IC and EC values were calculated and the plant extract (50 µL) at various concentrations. The 50 50 from linear regression analysis. The correlation coefficients reaction mixture was homogenized, and its absorbance was (R) between the total phenolic contents and the methods of recorded at 734 nm. Ethanol blanks were run in each assay, and antioxidant activity were demonstrated by employing EXCEL all measurements were done after at least 6 min. Similarly, the (2010). reaction mixture of standards group was obtained by mixing ·+ ABTS solution (950 µL) and BHT and Trolox (50 µL). The Results inhibition percentage of ABTS·+ radical was calculated using the following formula: In this study, the total extract yield, total phenolics, flavonoid ·+ ABTS scavenging effect (%) = [A0 – A1/A0] ×100 and tannin contents and the antioxidant activity of the aqueous where A0 and A1 have the same meaning as in Eq. (1). methanolic extracts of H. stoechas subsp. rupestre and P. saxatile As for the antiradical activity, the ABTS·+ scavenging subsp. saxatile collected from Algeria were determined. −1 ability was expressed as IC50 values (µg·mL ). Extraction yield, total phenolic, flavonoid, and condensed Chelating effect on ferrous ions tannin contents The ferrous ion chelating activity of the extracts was as- The method of extraction, based on liquid–solid ex-

– 417 – Farah Haddouchi, et al. / Chin J Nat Med, 2014, 12(6): 415−422 traction, was used to obtain the aqueous methanolic ex- highest extract yields with a higher yield for the flowers, tracts of the plants. The yield percentages obtained are unlike P. saxatile subsp. saxatile, whose yields were close shown in Table 1. H. stoechas subsp. rupestre had the for the two plant parts.

Table 1 Total antioxidant capacity, total polyphenol, flavonoid, and condensed tannin contents of aqueous methanolic extracts of H. stoechas subsp. rupestre and P. saxatile subsp. saxatile Yield Polyphenol Content Flavonoid content Tannin content Total antioxidant capacity

(%) (mg of GAE/g DW) (mg of CE/g DW) (mg of CE/g DW) (mg of GAE/g DW) FHS 18.70 ± 0.33 31.34 ± 1.92 10.48 ± 0.28 3.47 ± 0.21 97.50 ± 0.33 LSHS 13.50 ± 0.40 18.62 ± 0.41 09.10 ± 0.87 3.21 ± 0.19 29.43 ± 0.38 FPS 10.00 ± 0.13 04.80 ± 0.20 00.00 ± 0.00 00.9 ± 0.20 01.89 ± 0.12 LSPS 09.00 ± 0.33 18.82 ± 0.21 13.08 ± 0.28 2.08 ± 0.28 53.55 ± 0.36 FHS: Flowers of H. stoechas subsp. rupestre; LSHS: Leafy stems of H. stoechas subsp. rupestre; FPS: Flowers of P. saxatile subsp. saxatile; LSPS: Leafy stems of P. saxatile subsp. saxatile

In order to establish a relationship between the chemical 0.28 mg CE/g DW, respectively. content and the antioxidant activity, the total phenol, flavono- It has been reported that condensed tannins possess vari- id, and tannin contents of the aqueous methanolic extracts ous biological activities, such as antioxidant activity [24]. The were determined. The total phenolic contents of the extracts, content of condensed tannins was expressed with reference to as estimated by the Folin-Ciocalteau reagent method with the standard curve (Y = 0.000 76 x – 0.007 6). Levels of reference to the standard curve (Y = 0.002 53 x + 0.013 44), tannins are less important than flavonoids (Table 1). They are ranged from 4.80 ± 0.20 and 31.34 ± 1.92 mg GAE/g DW close in the extracts of leafy stem and flower of H. stoechas (Table 1). The highest level of phenolics was found in the subsp. rupestre (3.21 ± 0.19 and 3.47 ± 0.21 mg CE/g DW, extract of H. stoechas subsp. rupestre flower, while the lowest respectively). The extract of P. saxatile subsp. saxatile leafy level was in the extract of P. saxatile subsp. saxatile flowers. stem is richer in tannins than the flower extract (2.08 ± 0.28 Flavonoids are one of the most numerous and widespread and 0.9 ± 0.20 mg CE/g DW, respectively). group of phenolic compounds in higher plants [23]. The con- Antioxidant activity of aqueous methanolic extracts tent of flavonoids in the extracts was expressed with reference There are several methodologies widely used to to a standard curve (Y = 0.003 55 x + 0.099 03). The highest measure the antioxidant capacity of extracts. In this study, level of flavonoids was found in P. saxatile subsp. saxatile the total antioxidant activity, DPPH• and ABTS•+ radical leafy stem extract (13.08 ± 0.28 mg CE/g DW), while the scavenging assays, β-carotene bleaching method, ferric flowers are devoid of these compounds. The flavonoid con- reducing power and metal chelating activity were examined tents of extracts of H. stoechas subsp. rupestre leafy stem and in order to have an indication of antioxidant capacity of the flowers are close and correspond to 9.10 ± 0.87 and 10.48 ± test samples.

Table 2 Antioxidant activities, expressed as IC50 values for DPPH, ABTS, β-carotene, and iron chelating effect, and as EC50 value for iron reducing power, for the aqueous methanolic extracts and standards IC EC 50/DPPH 50/ Iron reducing power IC (µg·mL−1) IC (mg·mL−1) IC (µg·mL−1) (µg·mL−1) (µg·mL−1) 50/ABTS 50/Iron chelation 50/β-carotene FHS 6.57 ± 0.25 466.52 ± 10.50 94.54 ± 1.30 10.48 ± 0.10 121.81 ± 0.55 LSHS 8.72 ± 0.20 697.14 ± 5.50 337.71 ± 2.50 10.26 ± 0.15 191.46 ± 1.05 FPS 129.77 ± 0.55 3335.4 ± 16.95 n.t. n.t. n.t. LSPS 5.65 ± 0.10 355.24 ± 15.00 74.84 ± 1.05 04.62 ± 0.10 51.53 ± 0.40 99.64 ± 1.80 a 73.1 ± 1.70 a Standards 10.50 ± 0.40 a 0.0465 ± 0.0003 d 48 ± 0.90 e 53.67 ± 2.40 b 58.14 ± 2.50 c FHS: Flowers of H. stoechas subsp. rupestre; LSHS: Leafy stems of H. stoechas subsp. rupestre; FPS: Flowers of P. saxatile subsp. saxatile; LSPS: Leafy stems of P. saxatile subsp. saxatile. n.t.: not tested, a BHT, b Ascorbic acid, c Trolox, d EDTA, e BHA

Total antioxidant capacity capacity of extracts and levels of flavonoids (R = 0.64) and The phosphomolybdenum method is based on the formation tannins (R = 0.72), and a significant relationship between this of green Mo(V) complexes with a maximum absorption at 695 capacity and polyphenol content (R = 0.95). nm [17]. The results indicated that the aqueous methanolic extract DPPH radical-scavenging activity of H. stoechas subsp. rupestre flowers tested had strong total The DPPH assay has been widely used to provide basic antioxidant activity (97.5 ± 0.33 mg of GAE/g DW). The extract information on the antioxidant ability of plant extracts, be- of P. saxatile subsp. saxatile flowers has a very low activity (Ta- cause this method has shown to be rapid and simple [18]. The ble 1). Table 3 shows a correlation between the total antioxidant benefit of employing the DPPH assay is due to the high

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Table 3 Correlation coefficients (R) for relationships between assays and levels of polyphenols, flavonoids and tannins

TAC IC50/DPPH EC50/ Iron reducing power IC50/ABTS IC50/β-carotene EC50/Iron chelation Polyphenols 0.95 –0.83 –0.84 –0.45 –0.01 0.51 Flavonoids 0.64 –0.96 –0.98 –0.8 –0.98 –0.93 Tannins 0.72 –0.85 –0.82 0.4 0.76 0.99 stability of the DPPH radical and its commercial form being in aqueous and organic solvents over a large range of pH ready to use [25]. The free radical scavenging activity of the values, and the reaction time is lower than the DPPH assay [25]. aqueous methanolic extracts of H. stoechas subsp. rupestre Therefore, it was considered necessary to further assess the and P. saxatile subsp. saxatile, tested were determined extracts against the synthetic ABTS free radical. Trolox and through the DPPH method and the results are presented in BHT were the reagents used as standards.

Table 2. IC50 is a parameter widely used to measure antioxi- The scavenging effect of aqueous methanolic extracts dant activity. As the IC50 value of the extract decreases, the and standards on the ABTS radical, expressed as IC50 values free radical scavenging activity increases. The scavenging (Table 2), was in the following order: Trolox BHT and effect of aqueous methanolic extracts and standard (BHT) on leafy stems of P. saxatile subsp. saxatile flowers of H. the DPPH radical expressed as IC50 values was in the follow- stoechas subsp. rupestre leafy stems of H. stoechas subsp. ing order: leafy stems of P. saxatile subsp. saxatile (5.65 ± rupestre. The leafy stems of P. saxatile subsp. saxatile extract −1 0.10 µg·mL ), flowers of H. stoechas subsp. rupestre (6.57 ± exhibited interesting antioxidant activity (IC50 = 74.84 ± 1.05 −1 −1 −1 0.25 µg·mL ), leafy stems of H. stoechas subsp. rupestre µg·mL ) compared to BHT (IC50 = 73.1 ± 1.70 µg·mL ). (8.72 ± 0.20 µg·mL−1), BHT (10.5 ± 0.40 µg·mL−1), and flow- The establishment of a correlation shows a correlation −1 ers of P. saxatile subsp. saxatile (129.77 ± 0.55 µg·mL ) (Ta- between ABTS IC50 and the content of flavonoids (R = –0.8) ble 2). and a low correlation with polyphenols (R = –0.45) and Results reveal that H. stoechas subsp. rupestre extracts condensed tannins (R = –0.4) (Table 3). (flower and leafy stem) and P. saxatile subsp. saxatile leafy The same order of activity observed in the ABTS method stem extract have a stronger effect of scavenging free radical was found in the DPPH method, confirmed by the very good than the positive control (BHT), which is not the case for P. correlation (R = 0.97) between these two methods (Table 4). saxatile subsp. saxatile flower extract. However, the DPPH values are lower than those obtained Table 3 shows good correlations between the total from the ABTS assay. polyphenols, flavonoids, condensed tannin contents and DPPH Table 4 Correlation coefficients (R) for relationships between IC50 of extracts, with significant coefficients correlation R of assays –0.83, –0.96 and –0.85, respectively. Iron reducing DPPH β-Carotene Iron chelation Iron reducing power power Iron reducing The reducing capacity of the extract, another significant 0.99 1 power indicator of antioxidant activity, was also found to be appre- β-Carotene 0.97 0.98 1 ciable. Table 2 shows the reducing power potentials of the aqueous methanolic extracts of the test plants in comparison Iron chelation 0.70 0.72 0.85 1 with standards (ascorbic acid and BHT). The P. saxatile subsp. ABTS 0.97 0.97 0.90 0.53 saxatile leafy stem extract showed stronger reducing power −1 (EC50 = 355.24 ± 15.00 µg·mL ) than did other extracts, but Iron(II) chelation not stronger than the standards. P. saxatile subsp. saxatile The ability to chelate transition metals can be considered flower extract was clearly less important. This less significant as an important antioxidant mode of action. The IC50 values show activity confirms the result of the total antioxidant and the that the P. saxatile subsp. saxatile leafy stem extract had the DPPH radical-scavenging activities, and the antioxidant ac- highest ferric ion-chelating capacity (IC50 = 4.62 ± 0.10 tivity of P. saxatile subsp. saxatile flower extract was not mg·mL−1), followed by the extracts of flowers and leafy determined by the other methods. stem of H. stoechas subsp. rupestre (IC50 of 10.26 ± 0.15 The results showed a correlation between the levels of and 10.48 ± 0.10 mg·mL−1, respectively) (Table 2). None of total phenolic, flavonoids, and condensed tannins and the the aqueous methanolic extracts were as effective as the posi- −1 reducing power of the extracts with values of R equal to –0.84, tive control EDTA (IC50 = 0.046 ± 0.000 3 mg·mL ). The –0.98 and –0.82, respectively (Table 3). ferrous ion chelating abilities were highly correlated with the Scavenging ability of ABTS·+ flavonoid and tannin contents with values of R equal to –0.93 ABTS is another synthetic radical, and more versatile and 0.99, respectively. The data of the correlation (R) with than DPPH, because the ABTS model can assess the sca- total polyphenolic content was 0.51. venging activity for both the polar and non-polar samples [20], β-Carotene bleaching assay and has the advantage of the working solution being soluble Chelating activity and synthetic free radical scavenging

– 419 – Farah Haddouchi, et al. / Chin J Nat Med, 2014, 12(6): 415−422 models are valuable tools to assess the potential antioxidant antioxidant capacity and the ABTS methods. With the excep- activity of plant extracts. However, these systems do not use a tion of the extract of P. saxatile subsp. saxatile flowers, all food or biologically relevant, oxidizable substrate, so no di- extracts had an activity greater than that of BHT by the DPPH rect information on the protective effects of the plant extracts method and an activity similar to that of BHT and BHA by can be determined [26]. In this assay, the peroxyl radicals formed the ABTS and β-carotene methods. Evaluated by the iron when linoleic acid is oxidised attack the highly unsaturated reducing and chelating powers methods, all extracts have a β-carotene molecules that consequently undergo fast much lower activity than the standard compounds. discoloration [27]. Antioxidant activity, which was demonstrated There are a few previous studies on the antioxidant ca- by the ability of the samples to inhibit the bleaching of pacity of Phagnalon species. According Conforti et al [34], the β-carotene, was measured and compared to the positive methanolic extract of P. saxatile (L.) Cass., preceded by two control (BHA). As shown in Table 2, the aqueous methanolic successive extractions with petroleum ether and chloroform, extracts showed antioxidative activity expressed as IC50 val- showed significant activity by DPPH with an IC50 value of 25 ues, in the following order: BHA and leafy stems of P. sax- µg·mL−1, much less than ascorbic acid. The aqueous metha- atile subsp. saxatile flowers of H. stoechas subsp. rupestre nolic extract of P. saxatile subsp. saxatile leafy stems is much leafy stems of H. stoechas subsp. rupestre. The aqueous more important. methanolic extract of the leafy stems of P. saxatile subsp. The results of the in vitro antioxidant potential of extracts saxatile inhibited the oxidation of linoleic acid (IC50 = 51.53 ± of Helichrysum species showed that methanolic extracts of −1 −1 0.40 µg·mL ) compared to BHA (IC50 = 48 ± 0.90 µg·mL ). the sixteen Helichrysum species collected in Turkey had a The results of the relationship between inhibition of bleaching strong total antioxidant capacity expressed as mg of ascorbic of β-carotene and the phenolic compound contents of differ- acid equivalents (AAE)/g dry extract. H. noeanum was the ent extracts show a correlation between the flavonoids and con- most active [6]. H. monizii had the highest antioxidant poten- densed tannins with values of R equal to –0.98 and 0.76, respec- tial by the DPPH, ABTS, ferric reducing and β-carotene as- tively (Table 3). There is no correlation between this method says, due to the presence dicaffeoylquinic acids [25]. Some and the polyphenol contents (R = −0.01). species had comparable activity to that of the standard com- Correlation analysis between assays pound BHT, namely H. longifolium aqueous extract using The correlation analysis shows a very good correlation tests involving inhibition of superoxide anions, DPPH, and [35] −1 between DPPH, iron reducing power, ABTS, and β-carotene ABTS , H. stoechas subsp. barellieri (IC50 = 7.95 µg·mL ) (R between 0.9 and 0.99), and an average correlation between by the DPPH method [6], H. chionophilum, H. noeanum, H. these methods and the chelating power (R between 0.53 and plicatum subsp. plicatum, and H. arenarium subsp. aucheri, 0.85) (Table 4). in the β-carotene/linoleic acid test system [23]. H. stoechas from Italy had very high antioxidant activity comparable to Discussion that of Trolox and vitamin E, by radical scavenging method in [36] No work on the determination of the levels of phenolic cellular model . However, H. stoechas of Portugal has compounds was found on Phagnalon species, except for P. shown low activity in comparison with Trolox by DPPH (IC50 = [28] −1 −1 graecum , wherein the extraction was carried out succes- 520 µg·mL ), reducing power (IC50 = 140 ± 10 µg·mL ), and −1 sively by different solvents. Few references concerning the β-carotene bleaching inhibition (IC50 = 250 ± 80 µg·mL ) me- content of phenolic compounds of Helichrysum species could thods, respectively [32]. By the DPPH method, H. chasmolyci- [6, 29-31] −1 [37] be found , with contents that change from one sample cum extract (IC50 = 920 µg·mL ) , and methanolic extracts of −1 [6, 23] to another. The differences in phenolic compound contents of H. chionophilum (IC50 40 µg·mL ) showed less activity Helichrysum species may be due to the difference in their than the other Helichrysum species. When compared with chemical composition, extraction methods, collection time, those results, the other Helichrysum species showed less ac- collection area, and season. Some authors reported that phe- tivity than H. stoechas subsp. rupestre, except for H. stoechas nolic compounds are unstable and readily become non-antiox- subsp. barellieri from Turkey, by the DPPH method and H. idative under heating [32-33]. stoechas from Portugal, by reducing power. To completely elucidate a full profile of the antioxidant Since there are a large number of different types of anti- capacity of extracts, different antioxidant capacity assays oxidant compounds, it is not clear which components are (total antioxidant capacity, DPPH, iron reducing power, responsible for the observed antioxidant capacity. Some authors ABTS, β-carotene, and Iron (II) chelation) were used in this have shown that high total phenol content increases the antioxi- study. Of the four extracts examined, the antioxidant capacity dant activity [38], and the key role of phenolic compounds as sca- of P. saxatile subsp. saxatile leafy stem extract was signifi- vengers of free radicals is emphasized in several reports [39]. To cantly higher than other extracts assayed by the β-carotene explore the influence of the phytochemical constituents on and iron (II) chelation methods. However, there were no sig- antioxidant capacity, the correlation between the phenolic nificant differences in the extracts of the leafy stems and contents and antioxidant activity was determined. The anti- flowers of H. stoechas subsp. rupestre, except for the total oxidant capacity of the extracts appears to be largely influ-

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Cite this article as: Farah Haddouchi, Tarik Mohammed Chaouche, Riadh Ksouri, Faten Medini, Fatima Zohra Sekkal, Abdel- hafid Benmansour. Antioxidant activity profiling by spectrophotometric methods of aqueous methanolic extracts of Helichrysum stoechas subsp. rupestre and Phagnalon saxatile subsp. saxatile [J]. Chinese Journal of Natural Medicines, 2014, 12 (6): 415-422

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